In vitro safety signals for potential clinical development of the anti-inflammatory pregnane X receptor agonist FKK6.

Based on the mimicry of microbial metabolites, functionalized indoles were demonstrated as the ligands and agonists of the pregnane X receptor (PXR). The lead indole, FKK6, displayed PXR-dependent protective effects in DSS-induced colitis in mice and in vitro cytokine-treated intestinal organoid cultures. Here, we report on the initial in vitro pharmacological profiling of FKK6. FKK6-PXR interactions were characterized by hydrogen-deuterium exchange mass spectrometry. Screening FKK6 against potential cellular off-targets (G protein-coupled receptors, steroid and nuclear receptors, ion channels, and xenobiotic membrane transporters) revealed high PXR selectivity. FKK6 has poor aqueous solubility but was highly soluble in simulated gastric and intestinal fluids. A large fraction of FKK6 was bound to plasma proteins and chemically stable in plasma. The partition coefficient of FKK6 was 2.70, and FKK6 moderately partitioned into red blood cells. In Caco2 cells, FKK6 displayed high permeability (A-B: 22.8 × 10-6 cm.s-1) and no active efflux. These data are indicative of essentially complete in vivo absorption of FKK6. The data from human liver microsomes indicated that FKK6 is rapidly metabolized by cytochromes P450 (t1/2 5 min), notably by CYP3A4. Two oxidized FKK6 derivatives, including DC73 (N6-oxide) and DC97 (C19-phenol), were detected, and these metabolites had 5-7 × lower potency as PXR agonists than FKK6. This implies that despite high intestinal absorption, FKK6 is rapidly eliminated by the liver, and its PXR effects are predicted to be predominantly in the intestines. In conclusion, the PXR ligand and agonist FKK6 has a suitable pharmacological profile supporting its potential preclinical development.

On the Possible Effect of Phytic Acid (Myo-Inositol Hexaphosphoric Acid, IP6) on Cytochromes P450 and Systems of Xenobiotic Metabolism in Different Hepatic Models.

As compounds of natural origin enter human body, it is necessary to investigate their possible interactions with the metabolism of drugs and xenobiotics in general, namely with the cytochrome P450 (CYP) system. Phytic acid (myo-inositol hexaphosphoric acid, IP6) is mainly present in plants but is also an endogenous compound present in mammalian cells and tissues. It has been shown to exhibit protective effect in many pathological conditions. For this paper, its interaction with CYPs was studied using human liver microsomes, primary human hepatocytes, the HepG2 cell line, and molecular docking. Docking experiments and absorption spectra demonstrated the weak ability of IP6 to interact in the heme active site of CYP1A. Molecular docking suggested that IP6 preferentially binds to the protein surface, whereas binding to the active site of CYP1A2 was found to be less probable. Subsequently, we investigated the ability of IP6 to modulate the metabolism of xenobiotics for both the mRNA expression and enzymatic activity of CYP1A enzymes. Our findings revealed that IP6 can slightly modulate the mRNA levels and enzyme activity of CYP1A. However, thanks to the relatively weak interactions of IP6 with CYPs, the chances of the mechanisms of clinically important drug-drug interactions involving IP6 are low.

Effect of DSS-Induced Ulcerative Colitis and Butyrate on the Cytochrome P450 2A5: Contribution of the Microbiome.

Several studies have indicated the beneficial anti-inflammatory effect of butyrate in inflammatory bowel disease (IBD) therapy implying attempts to increase butyrate production in the gut through orally administered dietary supplementation. Through the gut-liver axis, however, butyrate may reach directly the liver and influence the drug-metabolizing ability of hepatic enzymes, and, indirectly, also the outcome of applied pharmacotherapy. The focus of our study was on the liver microsomal cytochrome P450 (CYP) 2A5, which is a mouse orthologue of human CYP2A6 responsible for metabolism of metronidazole, an antibiotic used to treat IBD. Our findings revealed that specific pathogen-free (SPF) and germ-free (GF) mice with dextran sulfate sodium (DSS)-induced colitis varied markedly in enzyme activity of CYP2A and responded differently to butyrate pre-treatment. A significant decrease (to 50%) of the CYP2A activity was observed in SPF mice with colitis; however, an administration of butyrate prior to DSS reversed this inhibition effect. This phenomenon was not observed in GF mice. The results highlight an important role of gut microbiota in the regulation of CYP2A under inflammatory conditions. Due to the role of CYP2A in metronidazole metabolism, this phenomenon may have an impact on the IBD therapy. Butyrate administration, hence, brings promising therapeutic potential for improving symptoms of gut inflammation; however, possible interactions with drug metabolism need to be further studied.

Gut microbiota metabolizes nabumetone in vitro: Consequences for its bioavailability in vivo in the rodents with altered gut microbiome.

1. The underlying microbial metabolic activity toward xenobiotics is among the least explored factors contributing to the inter-individual variability in drug response. 2. Here, we analyzed the effect of microbiota on a non-steroidal anti-inflammatory drug nabumetone. 3. First, we cultivated the drug with the selected gut commensal and probiotic bacteria under both aerobic and anaerobic conditions and analyzed its metabolites by high-performance liquid chromatography (HPLC) with UV detection. To analyze the effect of microbiota on nabumetone pharmacokinetics in vivo, we administered a single oral dose of nabumetone to rodents with intentionally altered gut microbiome - either rats treated for three days with the antibiotic imipenem or to germ-free mice. Plasma levels of its main active metabolite 6 methoxy-2-naphthylacetic acid (6-MNA) were analyzed at pre-specified time intervals using HPLC with UV/fluorescence detection. 4. We found that nabumetone is metabolized by bacteria to its non-active metabolites and that this effect is stronger under anaerobic conditions. Although in vivo, none of the pharmacokinetic parameters of 6-MNA was significantly altered, there was a clear trend towards an increase of the AUC, Cmax and t1/2 in rats with reduced microbiota and germ-free mice.

Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities.

1. The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically. 2. The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with Ki of 35.95 ± 6.96 and 60.56 ± 3.53 µmol/l and CYP3A4 (inhibited by genistein with Ki of 23.25 ± 5.85 µmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (Ki of 57.69 ± 2.36 µmol/l) and equol (Ki of 38.47 ± 2.32 µmol/l). 3. Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.

Influence of diet supplementation with green tea extract on drug-metabolizing enzymes in a mouse model of monosodium glutamate-induced obesity.

PURPOSE: Consumption of dietary supplements with green tea extract (GTE) is popular for weight management, but it may be accompanied by various side effects, including interactions with drugs. The aim of the present in vivo study was to evaluate the effect of defined GTE (Polyphenon 60) in three dosage schemes on insulin, leptin and drug-metabolizing enzymes in obese mice. METHODS: Experimental obesity was induced by repeated s.c. application of monosodium glutamate to newborn mice. Green tea extract was administered in three dosage schemes in chow diet. The plasmatic levels of insulin and leptin were assayed using enzyme-linked immunosorbent assay. Enzyme activities and mRNA expressions of drug-metabolizing enzymes (totally 13) were analyzed in liver and small intestine using spectrophotometric and HPLC assays and RT-PCR, respectively. RESULTS: GTE-treatment decreased insulin and leptin levels. Eleven enzymes were significantly affected by GTE-treatment. Long-term administration of 0.01% GTE caused increase in the activity and mRNA level of cytochrome P450 3A4 (CYP3A4) ortholog in the liver as well as in the small intestine. Interestingly, short-term overdose by GTE (0.1%) had more pronounced effects on enzyme activities and mRNA expressions than long-term overdose. CONCLUSIONS: GTE-mediated induction of CYP3A4 ortholog, the main drug-metabolizing enzyme, could result in decreased efficacy of simultaneously or subsequently administered drug in obese individuals.

Interaction of rocuronium with human liver cytochromes P450.

Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 µM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6ß-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed.

Acyclic nucleoside phosphonates: a study on cytochrome P450 gene expression.

1. Nucleotide analogues comprise an important class of drugs used in treatment of viral infections but also cancer. These drugs affect the structural integrity of DNA and activate different pathways and processes in the cell and may directly or indirectly influence the drug metabolizing system. Adefovir dipivoxil (AD) and tenofovir disoproxil (TD) are nucleotide analogues approved for the treatment of chronic hepatitis B and/or HIV/AIDS infection. 2. To evaluate the risk of their drug-drug interactions on the level of drug metabolism, an effect of both compounds on cytochromes P450 expression was studied using cDNA microarrays, real-time RT-PCR and immunoblotting. Mice were given intraperitoneally 25 mg/kg of AD or TD, respectively. As a positive control, a combination of prototypic cytochromes P450 (CYP) inducers, phenobarbital and ß-naphthoflavone was chosen. 3. The data obtained showed a significant CYP induction in the positive control group, but no clinically significant induction of CYP genes by AD or TD was observed. Our results support the evidence of safety of AD and TD with respect to drug-drug interactions based on enzyme induction. These findings are important as a plethora of new antivirals of different types are being tested and introduced to clinical practice, mostly to be used in combinations.

Effect of dihydromyricetin on benzo[a]pyrene activation in rats.

OBJECTIVES: Flavanol dihydromyricetin (DHM) has been shown to counteract acute ethanol (EtOH) intoxication and reduce excessive EtOH consumption. Since this flavonoid is being considered for human use, the in vivo study of DHM interactions with the cytochrome P450 (CYP) multienzyme system in the respect of metabolic activation of a model food-born carcinogen, benzo[a]pyrene (BaP), is of high importance. Flavonoids of known properties, alpha-naphthoflavone (ANF) and beta-naphthoflavone (BNF) were included into the study to compare their and DHM effects on BaP-DNA adduct formation. METH0 DS: The flavonoids were administered by oral gavage either 72 hrs prior or simultaneously with a single dose of BaP to experimental rats. The expression of CYP1A1/2 enzymes was examined based on the enzymatic activity with a marker substrate, 7-ethoxyresorufin, and on Western blots. The nuclease P1 version of the 32P-postlabeling assay was used to detect and quantify covalent DNA adducts formed by BaP. RESULTS: Treatment of rats with a single dose of DHM or ANF prior to or simultaneously with BaP did not produce an increase in levels of CYP1A1 and in formation of BaP-DNA adducts in liver. BNF, a known inducer of CYP1A1, showed a synergistic effect on BaP-mediated CYP1A1 induction and BaP activation in liver. Contrary to that, in small intestine the stimulatory effect of BNF on both parameters was not detected. Animal pre-treatment with DHM or ANF before BaP administration resulted in a significant elevation of BaP-DNA adducts, namely in the distal part of small intestine, while the CYP1A1 mediated 7-ethoxyresorufin-O-deethylation (EROD) was decreased markedly. It is important to note that under all regimens of animal treatment, DHM or ANF produced the higher inhibitory effect on the BaP-DNA adduct formation and BaP-induced EROD activity of CYP1A1 when administered simultaneously than sequentially with BaP. Our data show that DHM or ANF did not enhance the BaP-activation leading to BaP-mediated genotoxicity (the formation of BaP-DNA adducts) in rat liver, however, in small intestine the pretreatment of rats with these flavonoids may enhance BaP genotoxicity. CONCLUSIONS: The data indicate that the intake of DHM prior to or simultaneously with the administration of BaP may increase the risk of a BaP-induced tumorigenesis in small intestine.

Flexibility of human cytochrome P450 enzymes: molecular dynamics and spectroscopy reveal important function-related variations.

To gain more complete insight into flexibility and malleability of five forms of human liver cytochrome P450 enzymes, which play major roles in drug metabolism (CYPs 1A2, 2A6, 2C9, 2D6 and 3A4), we employed UV/VIS and resonance Raman spectroscopy in combination with all-atomic molecular dynamics simulations under normal and high pressure conditions (300 MPa). In general, the high pressure reduces the flexibility of CYPs, which become more dense and compact as their radii of gyration and temperature B-factors diminish. The flexibility of CYPs spans the regions, which are localized in solvent exposed loops. A considerable degree of flexibility is also observed at amino-acids making the pw2 and solvent channels, which are suggested to serve for substrate access and/or product release. The number of water molecules as well as the number of protein backbone atoms of the active site in close proximity of heme cofactor generally increases under high pressure. This finding provides new insights regarding the interpretation of pressure-related Soret band red shifts. Presented results also point towards considerable differences between the CYP forms studied: CYP2A6 and CYP1A2 have the least malleable active sites while those of CYP2D6, CYP2C9 and CYP3A4 have considerably greater degrees of flexibility or malleability. In addition, the number of water molecules in the active site cavity of CYP3A4 anomalously decreases under high pressure due to opening of the active site. These results correlate with the known substrate promiscuity of the respective CYP forms, with CYP3A4 displaying the highest substrate promiscuity, corresponding to the most open and malleable active site, whereas CYP1A2 and CYP2A6 show a high substrate-specificity and have a small and rigid active sites.

Rosuvastatin suppresses the liver microsomal CYP2C11 and CYP2C6 expression in male Wistar rats.

The aim was to investigate whether rosuvastatin affect rat cytochrome P450 (CYP) 2C11 and CYP2C6. CYP2C11 and CYP2C6 are considered as counterparts of human CYP2C9, which metabolizes many drugs including S-warfarin, diclofenac or ibuprofen. The male Wistar rats were fed standard laboratory diet (STD) or high cholesterol diet (HCD, 1% of cholesterol, 10% of lard fat) for 21 days. Rosuvastatin administration in STD (0.03% w/w) resulted in decreased mRNA expression of CYP2C11 as well as of CYP2C6 (here significant) and in a significant decrease of the respective protein expression as well as of the enzyme activity of both CYP2C forms. When rosuvastatin was administered in the HCD, the mRNA expression of both CYP2C forms was significantly lowered; the protein and activity parameters did not show significant changes. These results suggest that CYP2C11 as well as CYP2C6 expression and activity are negatively affected by rosuvastatin and may be modulated by high cholesterol high fat diet. Therefore, it should be taken into consideration that drugs metabolized by CYP2C9 in human could interact with rosuvastatin, as it has been already suggested for warfarin (rosuvastatin has increased its anticoagulant effect in human), and for telmisartan, sildenafil and glimepiride.

The influence of rosuvastatin on liver microsomal CYP2C6 in hereditary hypertriglyceridemic rat.

OBJECTIVES: The aim of this study was to investigate whether rosuvastatin affects expression and activity of rat CYP2C6. This cytochrome P450 is considered to be a counterpart of human CYP2C9, which metabolizes many drugs, including diclofenac, ibuprofen or warfarin. DESIGN: Male hereditary hypertriglyceridemic (HHTg) rats were fed standard laboratory diet (STD) or high cholesterol diet (HCD: STD + 1% of cholesterol w/w + 10% of lard fat w/w) for 21 days. A third group of rats were fed high a cholesterol diet with rosuvastatin added (0.03% w/w). Expression of CYP2C6 was measured in liver samples using real-time PCR (mRNA level) and Western blotting (protein level). Formation of diclofenac metabolites (typical enzyme activity of CYP2C6) was analyzed using HPLC with UV detection. RESULTS: Administration of rosuvastatin to HHTg rats resulted in significantly increased mRNA expression and enzyme activity in HCD-fed animals; changes of CYP2C6 protein were non-significant. These results suggest that CYP2C6 expression and activity are positively affected by rosuvastatin in hereditary hypertriglyceridemic rats after intake of HCD. CONCLUSION: The results presented open the possibility that in humans, rosuvastatin may affect the metabolism of many drugs by influencing expression and activity of CYP2C6 (counterpart of human CYP2C9). Further studies are needed to elucidate the effects of this statin on CYP2C9 in humans.

The role of cytochromes P450 in the metabolism of selected antidepressants and anxiolytics under psychological stress.

In today's modern society, it seems to be more and more challenging to cope with life stresses. The effect of psychological stress on emotional and physical health can be devastating, and increased stress is associated with increased rates of heart attack, hypertension, obesity, addiction, anxiety and depression. This review focuses on the possibility of an influence of psychological stress on the metabolism of selected antidepressants (TCAs, SSRIs, SNRIs, SARIs, NDRIs a MMAs) and anxiolytics (benzodiazepines and azapirone), as patients treated with antidepressants and/or anxiolytics can still suffer from psychological stress. Emphasis is placed on the drug metabolism mediated by the enzymes of Phase I, typically cytochromes P450 (CYPs), which are the major enzymes involved in drug metabolism, as the majority of psychoactive substances are metabolized by numerous CYPs (such as CYP1A2, CYP2B6, CYP2C19, CYP2C9, CYP2A6, CYP2D6, CYP3A4). As the data on the effect of stress on human enzymes are extremely rare, modulation of the efficacy and even regulation of the biotransformation pathways of drugs by psychological stress can be expected to play a significant role, as there is increasing evidence that stress can alter drug metabolism, hence there is a risk of less effective drug metabolism and increased side effects.

Rapid Determination of Metronidazole and 2-Hydroxymetronidazole in Murine Blood Plasma.

Metronidazole is a drug used to treat bacterial and protozoan infections. Nowadays, it is one of the most frequently prescribed drugs worldwide. The main aim of this paper is to present a rapid, reliable and simple high-performance liquid chromatography (HPLC) method to determine metronidazole along with its primary metabolite, 2-hydroxymetronidazole, in plasma or serum using paracetamol as an internal standard. A total of 100% methanol was used to denature plasma proteins. After centrifugation, the supernatant was evaporated under nitrogen flow. The samples were dissolved in the mobile phase and injected into a Li-Chrospher RP-18 column. A total of 10 mmol/L NaH2PO4: acetonitrile (90:10, v/v) solution with a flow rate of 1 mL/min was used as the mobile phase. Metronidazole and 2-hydroxymetronidazole were detected at two different wavelengths at 320 nm and 311 nm, respectively. The method is characterized by high precision (relative standard deviation % < 6). The method was used for the determination of metronidazole and 2-hydroxymetronidazole in murine blood using small amounts of plasma (≤100 µL).

Butyrate, a typical product of gut microbiome, affects function of the AhR gene, being a possible agent of crosstalk between gut microbiome, and hepatic drug metabolism.

Modulation of gut microbiome composition seems to be a promising therapeutic strategy for a wide range of pathologic states. However, these microbiota-targeted interventions may affect production of microbial metabolites, circulating factors in the gut-liver axis influencing hepatic drug metabolism with possible clinical relevance. Butyrate, a short-chain fatty acid produced through microbial fermentation of dietary fibers in the colon, has well established anti-inflammatory role in the intestine, while the effect of butyrate on the liver is unknown. In this study, we have evaluated the effect of butyrate on hepatic AhR activity and AhR-regulated gene expression. We have showed that AhR and its target genes were upregulated by butyrate in dose-dependent manner in HepG2-C3 as well as in primary human hepatocytes. The involvement of AhR has been proved using specific AhR antagonists and siRNA-mediated AhR silencing. Experiments with AhR reporter cells have shown that butyrate regulates the expression of AhR target genes by modulating the AhR activity. Our results suggest also epigenetic action by butyrate on AhR and its repressor (AHRR) presumably through mechanisms based on HDAC inhibition in the liver. Our results demonstrate that butyrate may influence the drug-metabolizing ability of liver enzymes e.g., through the interaction with AhR-dependent pathways.

Butyrate Treatment of DSS-Induced Ulcerative Colitis Affects the Hepatic Drug Metabolism in Mice.

The development of inflammatory bowel disease (IBD) is associated with alterations in the gut microbiota. There is currently no universal treatment for this disease, thus emphasizing the importance of developing innovative therapeutic approaches. Gut microbiome-derived metabolite butyrate with its well-known anti-inflammatory effect in the gut is a promising candidate. Due to increased intestinal permeability during IBD, butyrate may also reach the liver and influence liver physiology, including hepatic drug metabolism. To get an insight into this reason, the aim of this study was set to clarify not only the protective effects of the sodium butyrate (SB) administration on colonic inflammation but also the effects of SB on hepatic drug metabolism in experimental colitis induced by dextran sodium sulfate (DSS) in mice. It has been shown here that the butyrate pre-treatment can alleviate gut inflammation and reduce the leakiness of colonic epithelium by restoration of the assembly of tight-junction protein Zonula occludens-1 (ZO-1) in mice with DSS-induced colitis. In this article, butyrate along with inflammation has also been shown to affect the expression and enzyme activity of selected cytochromes P450 (CYPs) in the liver of mice. In this respect, CYP3A enzymes may be very sensitive to gut microbiome-targeted interventions, as significant changes in CYP3A expression and activity in response to DSS-induced colitis and/or butyrate treatment have also been observed. With regard to medications used in IBD and microbiota-targeted therapeutic approaches, it is important to deepen our knowledge of the effect of gut inflammation, and therapeutic interventions were followed concerning the ability of the organism to metabolize drugs. This gut-liver axis, mediated through inflammation as well as microbiome-derived metabolites, may affect the response to IBD therapy.

Interaction of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates with human liver microsomal cytochromes P450: comparison with free doxorubicin.

Interaction of nine forms of human hepatic cytochromes P450 (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) with two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-based doxorubicin (DOX) conjugates designed for passive tumor targeting was studied using pooled human microsomes. The compounds used in this study were two high-molecular-weight HPMA copolymers bearing doxorubicin attached to the polymeric carrier by 1) hydrazone bond enabling intracellular pH-controlled drug release or 2) amide bond through enzymatically cleavable tetrapeptide GlyPheLeuGly spacer. Both polymeric conjugates differing in mechanism of their antitumor activity and the free doxorubicin as the control were tested for potential inhibition activity. Among nine cytochrome P450 forms studied, no HPMA copolymer with bound DOX caused an inhibition of potential clinical significance. The extent of inhibition of enzymatic activities of the cytochrome P450 forms studied was negligible with the exception of CYP2B6 and was apparently caused by DOX as no inhibition was observed with polymers alone, and the extent of inhibition by the complex corresponded to this of the free DOX at the same concentration. In conclusion, the polymers and their conjugates with DOX seem to be relatively safe, at least in this respect, i.e., of inhibition of the liver microsomal drug-metabolizing enzymes.

In vitro interaction of a novel neutrophil growth factor with human liver microsomal cytochromes P450 and the contribution of UDP-glucuronosyltransferases to its metabolism.

Neutrophil growth factors (NGFs) stimulate neutrophil growth and survival. The first synthetic cytokinin-derived NGF was recently discovered and is a prospective drug owing to its potential use in anti-inflammatory therapy. The metabolism of some cytokinin-derived drugs (e.g. R-roscovitine, olomoucine II) has already been studied and it has been shown that they may give rise to drug-drug interactions. In this in vitro study, the interactions of the novel neutrophil growth factor NGF1568 with two of the main classes of human drug-metabolizing enzymes, cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs), were tested. Of the CYPs evaluated, NGF1568 was found to inhibit only CYP2C9, by an uncompetitive mechanism and with a K(i) value of 349 µM. Formation of a glucuronide of NGF1568 was detected by LC/MS/MS analysis after it was incubated with human liver microsomes and UDP-glucuronic acid. The human recombinant UGT1A9 enzyme (major liver expression) and UGT1A7, UGT1A8, UGT1A10 enzymes (expressed in gastrointestinal tract instead of liver) were found to be responsible for NGF1568 glucuronidation. These results show that interaction of NGF1568 with CYPs is not as important as it is in the case of the cytokinin CDK inhibitors R-roscovitine and olomoucine II, but the conjugation enzymes (UGTs) play a major role in its metabolism. Thus, possible interference of NGF1568 with metabolism of other coadministered drugs at least on level of liver, kidney or intestinal UGTs should be thoroughly considered.

Evidence for differences in regioselective and stereoselective glucuronidation of silybin diastereomers from milk thistle (Silybum marianum) by human UDP-glucuronosyltransferases.

The flavonolignan silybin, the main component of silymarin, extract from the seeds of Silybum marianum, is used mostly as a hepatoprotectant. Silybin is almost 1:1 mixture of two diastereomers A and B. The individual UDP-glucuronosyltransferases (UGTs) contributing to the metabolism of silybin diastereomers have not been identified yet. In this study, the contribution of UGTs to silybin metabolism was examined. The potential silybin metabolites were formed in vitro by incubating silybin (i) with the human liver microsomal fraction, (ii) with human hepatocytes and finally (iii) with 12 recombinant UGTs (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17). High-performance liquid chromatographic (HPLC) techniques with UV detection and additionally MS detection were used for metabolite identification. Hepatocytes and microsomes formed silybin A-7-O-ß-D-glucuronides, B-7-O-ß-D-glucuronides, A-20-O-ß-D-glucuronides and B-20-O-ß-D-glucuronides. With recombinant UGTs, the major role of the UGT1A1, 1A3, 1A8 and 1A10 enzymes but also of the UGT1A6, 1A7, 1A9, 2B7 and 2B15 in the stereoselective reactions leading to the respective silybin glucuronides was confirmed. UGT1A4, UGT2B4 and UGT2B17 did not participate in silybin glucuronidation. The predominant formation of 7-O-ß-D-glucuronides and the preferential glucuronidation of silybin B diastereomer in vitro by human UGTs were confirmed.

Effects of Lactobacillus casei on the expression and the activity of cytochromes P450 and on the CYP mRNA level in the intestine and the liver of male rats.

OBJECTIVES: The aim of the study was to find whether probiotic Lactobacillus casei influences the expression or the activity of cytochromes P450 (CYP) and whether it has an influence on the level of CYP mRNA in male rats. DESIGN: Live bacterial suspension of L. casei was administered orally (gavage) to healthy male Wistar rats daily for 7 days. Control group of rats was treated with the saline solution. Sections of the duodenum, jejunum, ileum, caecum and colon were dissected from each experimental animal. In all individual samples, the expression of selected CYPs was determined by Western blotting. The levels of expression of CYPs were also evaluated by mRNA using the real-time PCR method. RESULTS: There were changes observed in the expression of CYP enzymes and in the CYP mRNA levels along the intestine after application of L. casei. The expression of CYP1A1 enzyme was found to be decreased in the proximal part of the jejunum and colon, CYP1A1 mRNA level was decreased in the distal part of the jejunum, ileum and caecum. Thus, the changes in CYP1A1 protein or mRNA were observed along the intestine of male rats. Similarly, a decreased expression of the caecal CYP2E1 mRNA and of the duodenal CYP3A9 mRNA after treatment of rats with L. casei was found. CONCLUSION: Probiotic L. casei might be able to contribute to prevention against colorectal cancer by decreasing levels of certain forms of xenobiotic-metabolizing enzymes; moreover, in general, there is a possibility of interactions with concomitantly taken pharmacotherapeutic agents.